The cytotoxic effects of chromium (VI) have been linked to formation of reactive oxygen species via its redox activation. In this study we show that endothelial nitric oxide synthase (eNOS) can directly reduce Cr(VI) generating superoxide in this process. Incubations containing Cr(VI) (50 micromole) and eNOS (1.1 microgram), NADPH (0.1 millimole), DTPA (0.1 millimole) in HEPES buffer (50 millimole, pH 7.4) generated the ESR signal of the paramagnetic species Cr(V). Addition of the spin trap 5,5-diethoxyphosphoryl pyrroline N-oxide (DEPMPO) to the above system yielded DEPMPO-superoxide adduct which was abolished by SOD. Both superoxide and Cr(V) yields were increased upon activation of eNOS with Ca(2+)/CaM. Parallel experiments demonstrated that eNOS activity is inhibited by Cr(VI) in a concentration-dependent manner. The incubation of cultured endothelial cells with Cr(VI) resulted in an increased ICAM-1 expression, suggesting that Cr(VI) induces expression of adhesive molecules during its metabolism. These results suggest that an increased generation of superoxide combined with eNOS inhibition may play a major role in the cytotoxicity of Cr(VI) in vascular cells.